The aim is to understand how the E1A gene of adenovirus (Ad) is able to regulate transcription from the promoters of other viral and cellular genes in infected and transformed cells and how the E1A gene from tumorigenic Ad12 is able to block MHC class 1 transcription. Overwhelming evidence indicates that the larger of the two major E1A proteins (289R) efficiently mediates transcription stimulation (transactivation) by modulating the effective concentration of cellular factor required for transcription. The 289R protein contains an internal stretch of 46 amino acids, referred to as the unique region, which is not present the smaller 243R E1A protein. Our aim is to analyze Ad5 E1A genes with point mutations in the unique region to understand how the 289R interacts with transcription factors. A major effort will be placed on examing a novel E1A missense mutant, hr5. The hr5 E1A gene has two phenotypes; its 289R gene product fails to transactivate other viral promoters but inhibits the transactivating ability of the wt 289R protein. Hr5 E1A is thought to sequester a transcription factor used by wt E1A. This effect can be assayed by cotransfection of plasmids into Hela cells where hr5 E1A from stimulating transcription from early promoter driven test plasmids. In hr5 E1A, we will use site- directed mutagenesis to determine the stringency of the serine to asparagine substitution responsible for blocking wt E1A transactivation and deletion analysis to map the domains of the hr5 289R protein that are required for the inhibition. We will determine if hr5 E1A prevents wt E1A induced binding of specific factors to regulatory sequences upstream of early viral promoters and whether hr5 E1A blocks wt E1A induced transcription from polymerase lll promoters. In addition to hr5, we will determine if metal (e.g., Zn) binds to a concensus cysteine motif in the unique region of E1A and the effect of other point mutations in the unique region on E1A functions. Ad12 E1A mutants will be generated for the purpose of defining domains required for transactivation, repression of class 1 genes and tumorigenesis. Transgenic mice will be used to evaluate how Ad12 E1A in the germline effects MHC expression and tumorigenesis. Lastly we will begin to isolate genes that are differentially expressed between Ad12 and Ad5 transformed cells using subtractive cDNA hybridization. The long goal term goal is to determine the role of E1A in transactivation and repression of cellular genes reponsible for transformation and tumorigeneis.